Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

ABSTRACT

Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of pending U.S.divisional patent application entitled “Multimodality Imaging ofReporter Gene Expression Using a Novel Fusion Vector in living Cells andAnimals”, filed on Mar. 5, 2009 and assigned Ser. No. 12/398,352, whichis a divisional of the U.S. patent application filed on Aug. 15, 2006and assigned Ser. No. 10/548,146, now issued as U.S. Pat. No. 7,524,678issued Apr. 28, 2009, and which claimed the benefit of PCT/US04/07154filed Mar. 8, 2004, which claimed the benefit of U.S. provisional patentapplication 60/452,913, filed Mar. 7, 2003.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contractsCA092865, CA082214, CA086306 awarded by the National Institutes ofHealth and under contract DE-FC03-87ER60615 awarded by the Department ofEnergy. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Repetitive monitoring of reporter gene expression in intact livinganimals is crucial for many applications, including cell trafficking,gene therapy studies, and transgenic models (Ray, P., et al. (2001)Semin. Nucl. Med. 31, 321-330). Noninvasive, real-time analysis ofmolecular events in intact living mammals is an active area of currentresearch (See, e.g., Bremer, C. & Weissleder, R. (2001) Acad. Radiol. 8,15-23). Several imaging technologies and new reporter genes are beingstudied for noninvasive imaging and quantitation of gene expression inliving subjects. Some of the imaging modalities and established reportergenes include single photon emission computed tomography (SPECT) usingHerpes Simplex Virus Type I thymidine kinase HSV1-tk, Somatostatin Type2 receptor, and Sodium/Iodide Symporter as reporter genes. Positronemission tomography (PET) using HSV1-tk and Dopamine Type 2 Receptor asreporter genes, MRI with various reporter genes, and optical imagingapproaches with fluorescence and bioluminescent reporter genes have alsobeen studied. A detailed review of reporter gene approaches for use inliving subjects can be found in Ray et al., supra.

Noninvasive imaging of reporter gene expression using various imagingmodalities is playing an increasingly important role in definingmolecular events in the field of cancer biology, cell biology, and genetherapy. It is important to be able to image reporter gene expression inliving cells, animals, and humans using a single reporter construct. Asingle reporter gene would facilitate rapid translation of approachesdeveloped in cells to preclinical models and clinical applications. Todate, various methodologies exist that allow the imaging of reportergene expression in living cells and animals noninvasively andrepetitively. Confocal laser microscopy, two-photon laser microscopy,and several other techniques are available for real-time imaging of geneexpression at the single cell level using fluorescence (Piston, D. W.Imaging living cells and tissues by two-photon excitation microscopy.Trends Cell Biol., 9: 66-69, 1999; Jakobs, S., et al., EGFP and DsRedexpressing cultures of Escherichia coli imaged by confocal, two-photonand fluorescence lifetime microscopy. FEBS Lett., 479: 131-135, 2000).For reporter gene imaging in living subjects PET, single photon emissioncomputed tomography, magnetic resonance imaging, and optical imaging arewell standardized and are being used extensively in small animal models(Ray, P., et al. Monitoring gene therapy with reporter gene imaging,Semin. Nucl. Med., 31: 312-320, 2001; Wu, J. C., et al. Optical imagingof cardiac reporter gene expression in living rats, Circulation, 105:1631-1634, 2002; Bhaumik, S., and Gambhir, S. Optical imaging of renillaluciferase reporter gene expression in living mice, Proc. Natl. Acad.Sci. USA, 99: 377-382, 2002) and more recently with PET in humans(Yaghoubi, S. S., et al. PET imaging of FHBG in humans: a tracer formonitoring herpes simplex virus type 1 thymidine kinase suicide genetherapy, J. Nucl. Med., 41: 73P-74P, 2000; Jacobs, A., et al.,Positron-emission tomography of vector-mediated gene expression in humangene therapy for gliomas, Lancet, 358: 727-729, 2001). These imagingtechniques play important roles in defining critical pathways involvedin tumorigenesis, metastasis, and evaluating the efficiency of genetherapy strategies (Vooijs, M., et al., Noninvasive imaging ofspontaneous retinoblastoma pathway-dependent tumors in mice, CancerRes., 62: 1862-1867, 2002; Gambhir, S. S. Molecular Imaging of cancerwith positron emission tomography, Nat. Rev. Cancer, 2: 683-693, 2002,Yang, M., et al., Whole-body optical imaging of green fluorescentprotein expressing tumors and metastases. Proc. Natl. Acad. Sci. USA,97: 1206-1211, 2000). Each of these modalities has unique applications,advantages, and limitations that can be complementary to othermodalities. A cell-based technique is not useful for whole body in vivoimaging studies, whereas techniques involved in imaging at the tissue ororganism level do not have the resolution power to image gene expressionat the cellular level. Among the whole body imaging modalities, theradionuclide-based techniques have high sensitivity, good spatialresolution, and are tomographic in nature but are somewhat limited bytheir higher cost and especially for PET, the need for a cyclotron forproduction of isotopes for most tracers. In contrast, optical imagingtechniques (fluorescence and bioluminescence) represent a low cost andquick alternative for real-time analysis of gene expression in smallanimal models (Wu, supra; Yang, supra) but are limited by depthpenetration and cannot be easily generalized to human applications.

To overcome the shortcomings of each modality, a multimodality approachshould be very useful for detecting reporter gene expression. Combiningtwo or more different technologies (e.g., PET with optical) through aunified vector would have the advantage of speed and ease of validatingapproaches in small animals that in turn can be translated to humans.Such a vector might be achieved by several different approaches. Asingle reporter gene can be investigated for a single substrate doublylabeled with different signatures such as a radioactive nuclide(suitable for radionuclide imaging) or a nonradioactiveparamagnetic/bioluminescent/fluorescent molecule (suitable for magneticresonance or optical imaging) and thus can be imaged by differentimaging modalities. However, development of such substrates is oftendifficult because of the complex chemical nature of the biomolecules andlimitations on required pharmacokinetics in vivo.

What is needed, therefore, is a single vector harboring two or moredifferent reporter genes imaged by two or more different techniques(e.g., one radionuclide and one optical). Coexpression of multiple genesis generally achieved by using multiple promoters by insertion of aninternal ribosomal entry site or by fusing the two (or more) genes intoa single translational cassette (Ray, P., et al., supra, Semin. Nucl.Med., 31: 312-320). Our laboratory has successfully used tk (HSV1-sr39thymidine kinase, an improved PET reporter gene over the wild-typeHSV1-tk when using the guanosine analogues as tracer) and rl (renillaluciferase, a bioluminescence optical reporter gene) as separate imagingtools for studying the location, magnitude, and time variation ofreporter gene expression in living subjects (Bhaumik, S., supra;Gambhir, S. S., et al., A mutant herpes simplex virus type 1 thymidinekinase reporter gene shows improved sensitivity for imaging reportergene expression with positron emission tomography, Proc. Natl. Acad.Sci. USA, 97: 2785-2790, 2000; Yu, Y., et al., Quantification of targetgene expression by imaging reporter gene expression in living animals,Nat. Med., 6: 933-937, 2000).

The present inventors have now constructed and validated a novel tk andrl fusion protein imaged by microPET and bioluminescent optical CCDimaging modalities in tumor xenograft-bearing living mice. The presentinventors have further constructed and validated triple fusion reporterconstructs, including one combining a synthetic renilla luciferase(hrl), a red fluorescence protein (rfp or DsRed2) and a truncatedversion of sr39tk (ttk) that can be used to image in a single live cellusing a fluorescence microscope and in living mice with both an opticalcooled charged couple device (CCD) camera and microPET.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Measuring reporter gene expression using two different imagingmodalities when two reporter genes are simultaneously expressed from afusion vector. tk, a PET reporter gene is fused with rl (renillaluciferase), an optical bioluminescence reporter gene with the help of apolynucleotide coding for a short 20 a.a. long spacer and expression isdriven by a CMV promoter. Transcription of this fusion vector yields asingle mRNA, and subsequent translation leads to a single polypeptidethat is capable of retaining partial if not full activities of the twoproteins fused. Noninvasive, quantitative, and repeated imaging of thelocation and magnitude of the both reporter gene expression either bytrapping of a PET reporter probe (e.g., FHBG phosphorylated by TKenzyme) or by catalysis of an optical reporter probe (e.g., productionof light through RL-coelenterazine reaction) can be imaged by bothmicroPET and optical CCD camera in living subjects, respectively.

FIG. 2. Functional and biochemical characterization of TK-RL fusionproteins. A, TK and RL activity exhibited by the tk-rl fusion constructsin 293T (1) and N2a (2) cell lines. Cells were transiently transfectedwith tk20rl, tk18rl, tk10rl, CMVtk, CMV-rl, and CMV-β-gal and harvestedat 24 h and assayed for the TK and RL enzyme activities. Values for TKand RL activity were normalized with β-GAL activity for eachtransfection. The TK activity is expressed as (percentage of conversionof 8-³H— penciclovir to its phosphorylated form)/μg protein/min. The RLactivity is expressed as relative light units/μg protein. Error barsrepresent SE for triplicate measurements. B, Western blot analysis ofTK20RL fusion protein. Twenty μg of total cellular protein obtained fromthe cell lysates of transiently transfected 293T cells with tk20rl, tk,and rl plasmids were resolved in a 10% SDS polyacrylamide gel,transferred, and probed with (1) anti-TK and (2) anti-RL antibodies,respectively. Both the antibodies recognize the TK20RL fusion at ˜80 kDaand also a 36-kDa (by anti-RL antibody) and a 46-kDa (by anti-TKantibody) fragment (first lanes of both 1 and 2) as a result of partialcleavage. The polyclonal anti-TK antibody and monoclonal anti-RLantibody also recognize TK at ˜46 kDa (second lane from left in 1) andRL at ˜36-kDa (third lane from the left in 2), respectively.

FIG. 3. In vivo imaging of tk20rl fusion protein by using two differentmodalities. A, optical and microPET imaging of tk20rl fusion constructin the same nude mouse. A total of 2×10⁶ of N2a cells stably expressingthe tk20rl fusion construct and control N2a cells was implanted s.c. onleft and right shoulders in a nude mouse. After 4 days when each tumorwas ˜3-4 mm in diameter, the mouse was first scanned in the CCD cameraafter injection of coelenterazine via tail vein and bioluminescencesignal was recorded as maximum p/sec/cm2/sr (left panel). The same mousewas imaged 4 days later by microPET using FHBG (center panel) and againusing FDG on the following day (right panel). The tumor formed bytk20rl-expressing cells shows high bioluminescence as well as FHBGaccumulation in comparison to the control tumor. The FDG imagerepresents the viability of both tk20rl and control tumor. Nonspecificaccumulation of tracer was found in the gastrointestinal tracts (GI),bladder in case of FHBG (attributable to clearance of tracer), and in GItract and brain in case of FDG (attributable to high metabolicactivity). B, in vivo correlation of TK and RL gene expression exhibitedby four clones of N2a cells stably but differentially expressing thetk20rl fusion. A total of 2×10⁶ cells of each of four clones wereimplanted on the left shoulders of two nude mice each, and after 8-10days, mice were imaged by microPET and optical CCD camera on the sameday. Plot of % ID/g of FHBG versus bioluminescence signal as expressedas maximum (p/sec/cm²/sr) obtained from the ROI drawn on the tumors ofimages (R²=0.89). Each of the eight data point represents ROI data fromthe bioluminescent and PET image of the same mouse.

FIG. 4. Imaging serial increase in rl gene expression over time intumors stably expressing the tk20rl fusion. A total of 2×10⁶ of N2acells stably expressing tk20rl fusion gene and control N2a cells wasimplanted on the left and right shoulders, respectively, of a singlenude mouse and imaged daily using the optical CCD camera after injectionof coelenterazine. A gradual increase in bioluminescence was observed inthe tumor expressing tk20rl fusion over time but not in the controltumor.

FIG. 5. mrfp1, hRL, and tTK activity exhibited by 293T, N2a, and A375Mcell lines transiently transfected with the hrl-mrfp-ttk fusionconstruct. 293T (A), N2a (B), and A375M (C) cells were transientlycotransfected with CMV-β-gal and either CMV-hrl-mrfp-ttk, CMV-ttk,CMV-hr/, or CMVmrfp1; harvested 24 h later; and assayed for mRFP1expression (A. 1, B. 1, and C. 1; by fluorescence microscopy) andtTK/hRL enzyme activities (A. 2, B. 2, and C. 2). Bars for thefluorescence micrographs represent 100 μm. Values for tTK and hRLactivity were normalized with B-galactosidase activity for eachtransfection.

The tTK activity is expressed as (percentage of conversion of [8-³H]penciclovir to its phosphorylated form)/μ protein/min. The hRL activityis expressed as relative light units (RLU)/μg protein. Error barsrepresent SE for triplicate measurements. The slightly higher tTKactivity exhibited by the hRL-mRFP-tTK fusion protein in comparison withthe positive tTK protein is not statistically significant; however, thehRL activity of the hRLmRFP-tTK fusion protein is significantly lower(P<0.05) than the positive hRL protein.

FIG. 6. Biochemical and flow cytometric characterization of hrl-mrfp-ttkfusion reporter gene expression. A, Western blot analysis ofhRL-mRFP-tTK fusion protein. Twenty μ of total cellular protein obtainedfrom the cell lysates of transiently transfected 293T cells withhrl-mrfp-ttk, ttk, and hrl plasmids were resolved in a 10% SDSpolyacrylamide gel and transferred and probed with anti-TK (A.1) andanti-RL (A.2) antibodies. A 100-kDa band was specifically recognized bythe antibodies only from the hRL-mRFP-tTK fusion samples. The polyclonalanti-TK and the monoclonal anti-RL antibody recognize tTK (second lanein A.1) and hRL (third lane in A.2) at about 36 kDa, respectively. B,flow cytometry plot and enzymatic activities of the positive expressersof CS-larl-mrfp-ttk (lentiviral vector)-infected 293T cells. One million293T cells were infected with CS-hrl-mrfp-ttk vector and sorted withfluorescence-activated cell sorting with a filter at 585±42 nm bandsetting. Highly fluorescing cells (˜33%) that migrated to the P3 sector(B.1) were collected and further tested for tTK and hRL enzymeactivities (B.2). The sorted fraction showed higher tTK and hRLactivities than the unsorted population. The TK activity is expressed as(percentage of conversion of [8-³H] penciclovir to its phosphorylatedform)/μg protein/min. The RL activity is expressed as relative lightunits (RLU)/μg protein.

FIG. 7. Results of imaging living mice. A, fluorescence,bioluminescence, and micro-positron emission tomography (PET) imaging oflarl-mrfp-ttk expression in the same living nude mouse. Ten million 293Tcells transiently expressing the CMV-hrl-mrfp-ttk, CMV-ttk, CMV-mrfp1,and CMV-hrl plasmids were implanted s.c. at four sites on the ventralside of a nude mouse and imaged the next day forfluorescence/bioluminescence and PET using a cooled charge-coupleddevice (CCD) camera and microPET, respectively. Fluorescence imaging wasperformed by placing the mouse in a CCD camera for 1 s, and afluorescence image was acquired with a excitation filter at 500-550 nmand an emission filter at 575-650 nm. Cells expressing the fusion (A.1,a) and mrfp1 (A.1, c) genes showed fluorescence and the signal isrecorded as maximum photons/sec/cm²/sr (A.1). The same mouse was thenscanned in the CCD camera for bioluminescence after injection ofcoelenterazine via tail vein, and bioluminescence signal was found incells expressing the fusion (A.2, a) and hrl (A.2, d) and recorded asmaximum photons/sec/cm2/sr (A.2). After the optical scan, the same mousewas imaged by microPET using 9-(4-[¹⁸F] fluoro-3-hydroxymethylbutyl)guanine (FHBG). Cells expressing the fusion reporter gene (A.3, a andA.4, a) and ttk gene (A.3, b and A.4, b) showed FHBG accumulation incoronal section (A.3) and trans-axial section (A.4). Nonspecificaccumulation of tracer was found in the gastrointestinal tracts andbladder (attributable to clearance of FHBG; A.3). B, in vivo correlationof hrl, mrfp1, and ttk gene expression exhibited by four clones of 293Tcells stably but differentially expressing the hrl-mrfp-ttk fusion. Tenmillion cells of each clone were implanted on the axillary regions ofthe ventral side of three nude mice (two clones in each mouse), andafter 24 h, mice were imaged by the cooled CCD camera and microPET.Plots of percentage of ID/g versus bioluminescence [expressed as maximumphotons/second/centimeter²/steradian (p/s/cm²/sr); B.1], bioluminescenceversus fluorescence (both expressed as maximum p/s/cm²/sr; B.2), andpercentage of ID/g versus fluorescence (expressed as maximum p/s/cm²/sr;B.3) were obtained from the regions of interest drawn over the regionsof cell implantation. Each of the six data points of each plotrepresents region of interest data from the fluorescence,bioluminescence, and microPET images of the same living mouse, with atotal of three mice (2 points/mouse).

FIG. 8. Multimodality imaging of metastasis of A375M cells stablyexpressing the hrl-mrfp-ttk fusion reporter gene in living mice. A,bioluminescence imaging of a SCID mouse injected with A375M cellsexpressing the hrl-mrfp-ttk vector at day 0. 7×10⁵ A375M cells stablyexpressing the triple fusion were injected via tail-vein in a SCID mouseand two hours later imaged for bioluminescence signal followingtail-vein injection of coelenterazine. Prominent bioluminescence signalwas found from the region of both the lungs [1.3-1.5×10⁵ max(p/sec/cm²/sr)]. B, bioluminescence imaging of the same SCID mouse atday 40. At day 40, the same mouse was imaged and relatively highbioluminescence signal [2×10⁵ max (p/sec/cm²/sr)] was found from theleft lung region and moderate signal from the right lung region. A faintbioluminescence signal (5×10³ p/sec/cm²/sr) was also present from theright pelvic region. C, microPET imaging of the same SCID mouse at day40. Following a bioluminescence scan, the mouse was imaged in microPETusing FHBG. Shown is a thin coronal slice of ˜1-mm thickness. A strongsignal (˜0.78% ID/g) was present from the chest region (Ch) with lowersignal (0.35% ID/g) from the lung region. The stronger PET signal wasfound to be from a metastatic tumor present deep inside the body, asevident from the fluorescence photograph (8.E). Note the gallbladder(GB) retains FHBG so background signal from the GB is also seen in themicroPET images. D, light photograph of the same SCID mouse aftersacrifice and organ exposure (image has been modified by using AdobePhotoshop version 6). E, whole body fluorescence imaging of the sameSCID mouse. Fluorescing metastatic tumors were found in lung and chestregions that correspond with the bioluminescence and PET images.

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used herein are: PET, positron emission tomography;tk/TK, HSV1-sr39 thymidine kinase gene/protein; rl/RL, renillaluciferase gene/protein; β-gal/β-GAL, β-galactosidase gene/protein; CCD,cooled charge-coupled device; FHBG,9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl) guanine; FDG, 2-[¹⁸F]fluoro-2-deoxyglucose; N2a, neuro 2a; FBS, fetal bovine serum; PCV,penciclovir; CMV, cytomegalovirus; ROI, regions of interest; % ID/g,percentage of injected dose/gram; p/sec/cm²/sr,photons/second/cm²/steridian; gfp, green fluorescence protein; fl/FL,firefly luciferase gene/protein; rfp, red fluorescent protein; FACS,fluorescence-activated cell sorting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

One aspect of the present invention relates to a triple fusion vectorharboring a PET, a bioluminescence and a fluorescence reporter gene thatis imagable in living cells using a fluorescence microscope as well asin living mice using a Positron Emission Tomographic scanner, a chargecouple device (CCD) camera for both fluorescence and bioluminescenceimaging. This single reporter vector can be used to follow cellular andmolecular processes in living cells and subsequently in living subjects.By having a single reporter one can easily move between cells, smallanimal models and humans with great ease as compared to using separatereporters for each application. This is the first report of amultimodality reporter vector carrying three genes that cannoninvasively, repeatedly and quantitatively image gene expression inliving cells and in small living animals.

Another aspect of the present invention relates to a double fusionreporter construct carrying a PET reporter and a bioluminescent reportergene, which has been made and tested in various cell culture and inliving mice by tumor xenograft model.

In the practice of the present invention, a cell line or gene therapyvector or transgenic animal is marked with this reporter vector. Then,utilizing the three different imaging modalities (PET, Bioluminescenceand Fluorescence) and appropriate tracer/substrate or imagingconditions, gene expression is imaged in a single living cell as well asin living animals. The method can be used for detecting expression ofreporter genes in intact organisms, as well as in tissues and organsbeing maintained in culture or in tissue slices or other ex vivo or invitro situations.

In one embodiment of the present invention, the triple fusion vector,constructed using standard techniques, harbors HSV1-sr39 thymidinekinase (a PET reporter gene), renilla luciferase (a bioluminescent gene)and red fluorescence protein (a fluorescence gene). The vector may alsobe constructed with firefly luciferase and wild type HSV1 thymidinekinase genes. It is unique in that it can be used to track the cellularevents in a living cell by using a standard fluorescence microscope,which then can be translated in living subjects using the PET,bioluminescence and Fluorescence in vivo imaging modalities. This vectorhas the potential to translate approaches developed at the cellularlevel to pre-clinical models to clinical applications and has beentested in N2a (a murine neuroglial cell line) and 293T (human embryonickidney cell line) by transient transfection and is currently undervalidation in other cell lines. Clones of N2a cells stably expressingthis vector are also being isolated.

Reporter genes suitable for practice of the present invention include,but are not limited to, genes encoding polypeptides having imagableproperties; that is, polypeptides that may be detected by means such asMRI, PET, and visualization of bioluminescence or fluorescence, as wellas other means known in the art.

Polypeptides detectable by PET include, but are not limited to, wildtype and mutant thymidine kinase as described in the references citedherein (see, e.g., Yaghoubi, S. S., et al., J. Nucl. Med., 41: 73P-74P,2000; Jacobs, A., et al., Lancet, 358: 727-729, 2001).

Bioluminescent polypeptides include, but are not limited to, renillaluciferase and firefly luciferase. The enzyme renilla luciferase (RL),purified from sea pansy (Renilla reniformis), is a bioluminescentcompound that displays blue-green bioluminescence upon mechanicalstimulation. It is widely distributed among coelenterates, fishes,squids, and shrimps (J. W. Hastings, (1996) Gene 173, 5-11). It has beencloned and sequenced by Lorenz et al. (1991) Proc. Natl. Acad. Sci. USA88, 4438-4442, and used as marker of gene expression in bacteria, yeast,plant, and mammalian cells (W. W. Lorenz et al., (1996) J. Biolumin.Chemilumin. 11, 31-37). The enzyme RL catalyzes coelenterazine oxidationleading to bioluminescence.

Coelenterazine consists of an imidazolopyrazine structure,{2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazo[1,2-a]pyrazin-3-(7H)-one}that releases blue light across a broad range, peaking at 480 nm uponoxidation by RL in vitro (J. C. Matthews et al., (1977) Biochemistry 16,5217-5220).

Another well-characterized bioluminescent enzyme is known as fireflyluciferase (“FL”), because it was isolated from the lightning bug or thefirefly. Like RL, FL will also produce light in the presence of itssubstrate, which in this case is d-luciferin. FL has been used in liveanimal systems, and the resulting light production imaged. See, forexample, U.S. Pats. Nos. 5,650,135 and 6,217,847, the entire contents ofwhich are incorporated by reference herein. Firefly luciferase is a61-kDa single-subunit protein that catalyzes D-luciferin to produceoxyluciferin in the presence of oxygen, cofactors, Mg2+, and ATP toyield green light at 562 nm. The two luciferases (RL and FL) and the twosubstrates coelenterazine and D-luciferin are structurally unrelated.

Other examples of bioluminescent polypeptides are known in the art. Oneof skill in the art will appreciate that, depending on thebioluminescent polypeptide incorporated into the fusion vector, asuitable substrate may be administered to the cell (s) or animalcontaining the vector; e.g., coelenterazine for RL, D-luciferin for FL,and the like.

Examples of fluorescent polypeptides suitable for use in the presentinvention include, but are not limited to, red fluorescing protein andgreen fluorescing protein as described in the references cited herein(see, e.g., Yang, M., et al., Proc. Natl. Acad. Sci. USA, 97: 1206-1211,2000; Jakobs, S., et al., FEBS Lett., 479: 131-135, 2000).

It should be noted that incorporating fluorescent reporter genes intothe fusion vectors of the present invention allows for cell sortingusing FACS (Fluorescent activated cell sorting). This provides ahigh-throughput method for sorting cells expressing the fusion vector(and thus expressing the other marker genes present in the fusionvector).

In practice of the present invention, nucleic acid sequences encodingimagable polypeptides are inserted into an expression vector in frameand operably linked to an expression control sequence, so thatexpression of nucleic acid sequences produces a fused polypeptidecomprising two or more imagable polypeptides.

Expression vectors suitable for practice of the present invention arewell known in the art and depend, in part, on the cell type in whichexpression will be monitored. Examples of suitable vector include, butare not limited to pcDNA3.1 and R-Luc N fusion vector (BiosignalPackard, Canada). Others will be apparent to those of skill in the art.

The DNA sequences in the expression vector are operatively linked to anappropriate expression control sequence (s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses.

The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression, such asenhancer sequences. Where expression of a particular gene is ofinterest, expression control sequences for that gene of interest may beincorporated into the expression vector, as will be appreciated by theskilled artisan.

Nucleic acid sequences for several suitable reporter polypeptides arewell known in the art and are generally commercially available, asindicated herein and in the references cited herein.

Techniques for inserting nucleic acid sequences into expression vectors,such that nucleic acid sequences are operably linked to expressioncontrol sequences and are in frame, are widely known in the art. See,e.g., Sambrook, J., Fritsch, E. and Maniatis, T., Molecular Cloning: ALaboratory Approach, Second Edition (1989; Cold Springs HarborLaboratory Press).

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees centigrade; andpressure is at or near atmospheric.

Example 1 Double Fusion Reporter Introduction

Noninvasive imaging of reporter gene expression using various imagingmodalities is playing an increasingly important role in definingmolecular events in the field of cancer biology, cell biology, and genetherapy.

In this study, a novel reporter vector was constructed encoding a fusionprotein comprised of a mutant herpes simplex virus type 1 thymidinekinase (HSV1-sr39tk) (tk), a positron emission tomography (PET) reportergene, and renilla luciferase (rl), a bioluminescence optical reportergene joined by a 20 amino acid long spacer sequence. We validated theactivity of the two enzymes encoded by the fusion protein (tk20rl) incell culture. Then, tumors stably expressing the tk20rl fusion gene wereimaged both by microPET and optically using a cooled charge coupleddevice camera in xenograft-bearing living mice. Using a single fusionreporter (PET/optical) gene should accelerate the validation of reportergene approaches developed in cell culture for translation intopreclinical and clinical models. See FIG. 1 for an overview of thisapproach.

Materials and Methods.

Chemicals. FDG was synthesized at University of California at LosAngeles as described previously (Hamacher, K., Coenen, H. H., andStocklin, G. Efficient stereospecific synthesis of no-carrier-added 2[¹⁸]-fluoro-2-deoxy-D-glucose using amino polyether supportednucleophilic substitution. J. Nucl. Med., 27: 235-238, 1986). 8-³H—Penciclovir was obtained from Moravek Biochemicals (Brea, Calif.). FHBG(9-(4-[¹⁸F] fluoro-3-hydroxymethylbutyl) guanine) was also synthesizedat University of California at Los Angeles as detailed previously(Yaghoubi, S. S., Goldman, R., Barrio, J. R., Namavari, M., Iyer, M.,Satyamurthy, N., Herschman, H. R., Phelps, M. E., and Gambhir, S. S. PETimaging of FHBG in humans: a tracer for monitoring herpes simplex virustype 1 thymidine kinase suicide gene therapy. J. Nucl. Med., 41:73P-74P, 2000). Coelenterazine was purchased from Biotium, Inc.(Hayward, Calif.). The polyclonal anti-TK antibody was a kind gift ofDr. M. Black (Washington State University, Pullman, Wash.), and themonoclonal anti-RL antibody was purchased from Chemicon International(Temecula, Calif.).

Construction of tk-rl Fusion Gene. PCR amplification and standardcloning techniques were used to insert the tk gene from plasmid pcDNA3.1, HSV1-sr39tk (Yu, Y., Annala, A. J., Barrio, J. R., Toyokuni, T.,Satyamurthy, N., Namavari, M., Cherry, S. R., Phelps, M. E., Herschman,H. R., and Gambhir, S. S. Quantification of target gene expression byimaging reporter gene expression in living animals. Nat. Med., 6:933-937, 2000) in frame with the rl gene into the R-Luc N fusion vectors(Biosignal Packard, Canada). For PCR amplification, three different 3′end primers:

5′-GAGCCTCGAGGTTAGCCTCCCCCAT-3′ (SEQ ID NO: 1);

5′-GAGCGAATTCGTTAGCCTCCCCCAT-3′ (SEQ ID NO: 2); and

5′-GAGCAAGCTTGTTAGCCTCCCCCAT-3′ (SEQ ID NO: 3) were used along with thesame 5′ end primer:

5′-GCAGCTAGCCGCCACCATGGCTTCGTACCCC-3′ (SEQ ID NO: 4)

to eliminate the stop codon of the tk gene and introduce differentrestriction sites. Cloning of these three different PCR products of tkgene into three subtypes of R-Luc N fusion vector (N1, N2, and N3 thatdiffer from each other by 1 or 2 bases in their multi-cloning sites toprovide alternate reading frames) generated spacers differing in length,sequence, and composition.

Cell Lines, Transfection Procedures, and Stable Clone Isolation. C6 ratglioma cells (obtained from Dr. M. Black), N2a neuronal cell lines(obtained from Dr. Vincent Mauro, Scripps Research Institute, La Jolla,Calif.), and 293T human embryonic kidney cells (American Type CultureCollection, Manassas, Va.) were used. The C6 cells were cultured in highglucose, deficient minimal Eagle's medium supplemented with 5% FBS and1% penicillin (100 μg/ml), streptomycin (292 μg/ml), glutamine (100 mM),and histidinol (27 μg/ml) by volume. The N2a cells were cultured in highglucose DMEM supplemented with 10% FBS and 1% penicillin (100 μg/ml),streptomycin (292 mg/ml), and 293T cells were grown in MEM supplementedwith 10% FBS and 1% penicillin/streptomycin solution. All transient andstable transfections were carried out using the Qiagen Superfecttransfection reagent (Qiagen, Valencia, Calif.) following the protocolrecommended by the manufacturer. The N2a stable cell lines carrying thefusion gene construct were selected with 200 μg/ml of G418. The cloneswere initially screened for renilla luciferase activity using a CCDcamera (Bhaumik, S., and Gambhir, S. Optical imaging of renillaluciferase reporter gene expression in living mice. Proc. Natl. Acad.Sci. USA, 99: 377-382, 2002) and then assayed for thymidine kinaseactivity (Gambhir, S. S., Barrio, J. R., Phelps, M. E., Iyer, M.,Namavari, M., Satyamurthy, N., Wu, L., Green, L. A., Bauer, E.,MacLaren, D. C., Nguyen, K., Berk, A. J., Cherry, S. R., and Herschman,H. R. Imaging adenoviral-directed reporter gene expression in livinganimals with positron emission tomography. Proc. Natl. Acad. Sci. USA,96: 2333-2338, 1999).

TK, RL, and p-Gal Activity. Thymidine kinase activity assays wereperformed as previously described (Gambhir, S. S., supra, Proc. Natl.Acad. Sci. USA, 96: 2333-2338), and β-gal and renilla luciferase assayswere done using the (β-Gal enzyme assay system and Dual-LuciferaseReporter Assay System from Promega (Madison, Wis.), respectively.

Western Blot Analysis. The expression of TK and RL were evaluated byWestern blotting with a rabbit polyclonal anti-TK antiserum and a mousemonoclonal antirenilla antibody using cell lysates prepared from 293Tcells transfected with tk20rl, HSV1-sr39tk, or rl plasmids (Gambhir, S.S., Bauer, E., Black, M. E., Liang, Q., Kokoris, M. S., Barrio, J. R.,Iyer, M., Namavari, M., Phelps, M. E., and Herschman, H. R. A mutantherpes simplex virus type 1 thymidine kinase reporter gene showsimproved sensitivity for imaging reporter gene expression with positronemission tomography. Proc. Natl. Acad. Sci. USA, 97: 2785-2790, 2000). Asemiquantitative analysis of the Western blot was performed using theMacBAS V2.4 software (Fuji Base 5000, Tokyo, Japan).

MicroPET Imaging of Mice. Animal care and euthanasia were performed withthe approval of the University of California Animal Research Committee.Twelve- to 14-week old male nude mice (nu/nu) were injected s.c.with—2×10⁶ of N2a cells stably expressing the tk20rl fusion and controlnonexpressing N2a cells, and after 8-10 days, tumor-bearing mice werescanned in microPET as described earlier (Gambhir, S. S., supra, Proc.Natl. Acad. Sci. USA, 97: 2785-2790). The microPET images werereconstructed by using three-dimensional filtered back projection and aniterative maximum a posteriori algorithm (Qi, J., Leahy, R. M., Cherry,S. R., Chatziioannou, A., and Farquhar, T. H. High-resolution 3DBayesian image reconstruction using the microPET small-animal scanner.Phys. Med. Biol., 43: 1001-1013, 1998). ROI were drawn over the tumorarea. The ROI counts were converted to the % ID/g tumor using filteredback projection as previously described (Gambhir, S. S., supra, Proc.Natl. Acad. Sci. USA, 97: 2785-2790), and images shown werereconstructed with maximum a posteriori algorithm.

Optical Imaging of Renilla Luciferase Expression in Vivo. For in vivooptical imaging, mice implanted with stably expressing tk20rl fusionN2a, and control N2a cells were anesthetized and each mouse was theninjected with 10 μl of coelenterazine (stock solution, 2 μg/μl inmethanol) diluted in 90 μl of PBS (pH 7) via tail vein. Each animal wasthen placed supine in a light tight chamber, and whole body images wereobtained and quantified as described previously (Bhaumik, S., andGambhir, S., supra, Proc. Natl. Acad. Sci. USA, 99: 377-382).

Results

A HSV1-sr39 Thymidine Kiase PET and Renilla Luciferase BioluminescenceReporter Gene Fusion Vector Bearing the Coding Sequence for a 20 a.a.Long Spacer Maintains the Highest TK and RL Activity in Three DifferentCell Lines. We first constructed a fusion gene vector caring tk and rlreporter genes using the spacer lengths. The PCR-amplified tk genefragments from the pcDNA3.1-HSV1-sr39tk plasmid (Yu, Y., supra, Nat.Med., 6: 933-937) were cloned in frame into R-Luc-N fusion vectors togenerate a fusion gene construct under the CMV promoter. PCRamplification of tk gene using three different 3′ end primers andsubsequent cloning of these amplified fragments generated three tk-rlfusion constructs bearing spacers with length and sequence as indicated:

tk20rl (NSHASAGYQACGTAGPGSTG); (SEQ ID NO: 5)tk18rl (SRVCRISSLRYRGPGITG); (SEQ ID NO: 6) and tkl0rl (AVPRARDPTG).(SEQ ID NO: 7)

Plasmid DNA prepared from four to five clones for each spacer type weretransiently transfected in 293T cells and assayed for TK and RLactivity. The plasmid clones exhibiting the highest TK and RL activitieswere selected for additional studies (data not shown). Each of the threetk-rl fusion constructs were then subsequently cloned in pcDNA3.1 (+)backbone to directly compare results of each fusion with thepcDNA3.1-HSV1-sr39tk and pcDNA3.1-rl [rl was also cloned in pcDNA3.1 (+)from the R-Luc-N fusion vector], the positive control plasmids.

To compare the levels of reporter gene expression of each tk-rl fusionplasmids, three different cell lines [293T (FIG. 2A.1), N2a (FIG. 2A.2),and C6 (data not shown)] were transiently transfected with the threeplasmids (tk20rl, tk18rl, or tk10rl) along with positive controls (pcDNA3.1-tk and pcDNA3.1-rl) and negative controls (control cells mocktransfected). Each cell line was also cotransfected with the (3-galreporter gene to normalize for transfection efficiency. After 24 h, theexpression levels of all of the three reporter genes were assayed fromthe same cell lysates and TK and RL activities were normalized to β-GALactivity. Despite decreased TK enzyme activity seen by all of the fusionconstructs in comparison to the positive control (pcDNA3.1-HSV1-sr39tk),a trend of increase in the level of TK activity with increasing spacerlength is observed. The tk20rl plasmid (longest spacer) shows thehighest TK activity, which is still 45% (293T; FIG. 2A.1), 19% (N2a;FIG. 2A.2), or 22% (C6; data not shown) of that of the positive control.Interestingly, the RL activity of each construct is relatively higher(˜6-8 fold; FIG. 2, A.1 and A.2) than the positive control (pcDNA3.1-rl)and also increases with increasing spacer length.

Western Blot Analysis of Extracts from Cells Transiently Transfectedwith tk20rl Probed with anti-TK and anti-RL Antibodies Reveals thePresence of an 80-kDa Fragment, the Predicted Size of the TK20RL Fusion.To investigate tk20rl fusion reporter gene expression at the proteinlevel, cell lysates from tk20rl-, tk-, and rl-transfected 293T cellswere resolved by 10% SDS-PAGE and analyzed on Western blots by usingantibodies specific for TK or RL (FIG. 2B). The predominant bandrecognized by both anti-TK and anti-RL antibodies is of 80 kDa, theexpected size of the TK20RL fusion protein. However, in both cases, aweak band of similar size of either TK or RL alone when probed withrespective antibodies is seen. This is likely attributable to partialcleavage of the fusion protein. TK and RL proteins were recognized at ˜6and 36 kDa band by their specific antibodies. A semiquantitativeanalysis of the Western blot revealed that ˜36 and 25% of the totalfusion protein was cleaved into its TK and RL components, respectively.The lower molecular weight bands present in the positive TK sample mighthave resulted from partial degradation of the sample or nonspecificbinding of the polyclonal anti-TK antibody to other cellular proteins.

N2a Cells Stably Expressing the tk20rl Fusion Reporter Gene Can BeImaged in Living Mice Using a Tumor Xenograft Model by both microPET andOptical-cooled CCD Imaging Systems. In order to test the efficacy of afusion reporter vector for imaging of reporter gene expressionquantitatively and repeatedly in living subjects using two differentmodalities, we isolated several clones of N2a cells stably expressingtk20rl fusion gene and one exhibiting the highest TK, and RL activity,and tested for its ability to be imaged in vivo using microPET and acooled CCD camera in a tumor xenograft model. Five nude mice receiveds.c. injections in each shoulder with control N2a cells or tk20rlexpressing cells. When the tumors attained a minimum of 0.6-0.7 cm indiameter, mice were first scanned using the cooled CCD camera followedby a microPET scan. Optical imaging of these mice after tail-veininjection of coelenterazine reveal that the tumors expressing the tk20rlfusion show relatively high bioluminescence of ˜3081×10³+725×10³ maximum(p/sec/cm²/sr) in comparison to the control N2a tumors [˜3.1×10³+0.7×10³maximum (p/sec/cm²/sr), P<0.002; FIG. 3A]. Next, we scanned these miceby microPET using FHBG and finally on the following day using FDG. Wequantified the signal from each tumor directly from the microPET imagesto determine the % ID/g for FDG and FHBG. This % ID/g is a measure ofthe amount of tracer accumulated in a given tissue site normalized tothe injected amount and to the mass of the tissue examined. The FHBGaccumulation in the tumors reflects the TK activity of thetk20rl-expressing cells, whereas the FDG accumulation reflects themetabolic activity of the tumor cells. The mean % ID/g value for PHBGaccumulation in the tk20rl-expressing tumors (0.812+0.16) wassignificantly higher than the control N2a tumors (0.075+0.011; P<0.002)for the five mice. The mean FDG % ID/g values of tk20rl-expressing and-control tumors were not significantly different as expected (2.45±0.25versus 2.60±18; FIG. 3A). Although the cell culture data showed adecrease in TK activity with the tk20rl fusion in comparison toHSV1-sr39tk, microPET imaging reveals easily detectable FHBGaccumulation in the tumors expressing the TK20RL fusion protein.

We next examined whether tk and rl expression was correlated from thetk20rl fusion construct in living mice. Four clones of N2a cells stablybut differentially expressing the fusion vector were implanted as tumorsin eight nude mice (two mice for each clone) and microPET and opticalCCD imaging of those mice were performed on the same day. The % ID/gvalues for FHBG and the bioluminescence signal of the tk20rl expressingtumors were well correlated (R²=0.89) across the eight mice (FIG. 4B).The TK and RL activities of these four stable clones in vitro were alsowell correlated (R²=0.91; data not shown).

Renilla Luciferase Reporter Gene Expression Can Be Serially Measured inCells Stably Expressing the tk20rl Fusion Construct in Living Mice withHigh Sensitivity. One of the greatest advantages of opticalbioluminescence imaging is its comparatively high sensitivity (allowingdetection of low cell numbers) for imaging gene expression, whereasmicroPET imaging requires a greater tumor volume (3-5 mm) or mass ofcells to obtain a detectable signal. We therefore implantedtk20rl-expressing and -nonexpressing control N2a cells as tumors on boththe shoulders of four nude mice and imaged them daily using a cooled CCDcamera to monitor the expression level of the fusion reporter construct.Significant signal is not seen on the first day (both the control N2aand tk20rl-expressing tumors showed bioluminescence value maximum at˜4×10³ p/sec/cm²/sr), but after the second day, the optical signal inthe tk20rl-expressing tumors started increasing progressively andreached a maximum of 6×10⁶+1.5×10⁶ (p/sec/cm²/sr) on day 10, whereas thesignal in control tumors remained unchanged (12.0×10³+4.2×10³ maximump/sec/cm²/sr) throughout the study (FIG. 4). With the gradual increasein rl expression, we observed gradual growth of the tumors that attaineda diameter of ˜0.6-0.8 cm at day 10. We also attempted microPET imagingof these mice when the tumors were not palpable but were unable toobtain any detectable level of signal (data not shown). Presence of thebioluminescence rl reporter gene in the tk20rl fusion construct,therefore, confers a highly sensitive tool for monitoring reporter geneexpression.

Discussion

The construction of a novel fusion gene vector is described, the vectorharboring HSV1-sr39 thymidine kinase, a PET reporter gene and renillaluciferase, a bioluminescence reporter gene and its application inliving mice using two different imaging modalities is validated.HSV1-thymidine kinase (both wild-type and the sr39 mutant) arewell-established PET reporter genes (Jacobs, A., Voges, J., Reszka, R.,Lercher, M., Gossmann, A., Kracht, L., Kaestle, C., Wagner, R.,Wienhard, K., and Heiss, W. D. Positron-emission tomography ofvector-mediated gene expression in gene therapy for gliomas. Lancet,358: 727-729, 2001; Gambhir, S. S., supra, Proc. Natl. Acad. Sci. USA,97: 2785-2790; Yu, Y., supra, Nat. Med., 6: 933-937) for imaging geneexpression. Fusion vectors harboring wild-type tk and green gfp (tk-gfp)constructed by several groups (Wahlfors, J., Loimas, S., Pasanen, T.,and Hakkarainen, T. Green fluorescent protein (GFP) fusion constructs ingene therapy research. Histochem. Cell Biol., 115: 59-65, 2001; Jacobs,A., Dubrovin, M., Hewett, J., Sena-Esteves, M., Tan, C. W., Slack, M.,Sadelain, M., Breakefield, X. O., and Tjuvajev, J. G. Functionalcoexpression of HSV-1 thymidine kinase and green fluorescent protein:implications for noninvasive imaging of transgene expression. Neoplasia,1: 154-161, 1999) could retain sufficient levels of TK activity and beimaged by microPET. As shown herein, the sr39thymidine kinase-renillaluciferase fusion vectors constructed by us could also maintain the TKand RL activities in different cell lines at different levels. However,our attempt to build a fusion construct of tk and fl yielded a poorlyactive fusion protein (unpublished data). Another triple fusionconstruct bearing wild-type tk, fl, and neomycin genes (tk-fl-neo;Strathdee, C. A., McLeod, M. R., and Underhill, T. M. Dominant positiveand negative selection using luciferase, green fluorescent protein andbeta-galactosidase reporter gene fusions. Biotechniques, 28: 210-212,214, 2000) also showed very low TK activity in comparison to the tkvector alone in cell culture in our hands (unpublished data). Therefore,the nature of the fusion partner also affects the activity of the TKenzyme. Moreover the length of the spacer between the two proteins seemsto play an important role in maintaining functionality of each protein,which has also previously been reported for various fusion constructs(Wahlfors, J., supra, Histochem. Cell Biol., 115: 59-65). In addition,it is likely that the amino acid (a.a.) sequence and composition of thespacer can influence the activities of either enzyme. The order of thefusion partners of the construct might also influence the activities ofthe proteins depending on the positioning of critical amino acids in theprotein backbones. It has recently been shown that small changes inHSV1-tk, including several critical amino acids at the COOH-terminal end(Saijo, M., Suzutani, T., Niikura, M., Morikawa, S., and Kurane, I.Importance of C-terminus of herpes simplex virus type 1 thymidine kinasefor maintaining thymidine kinase and acyclovir-phosphorylationactivities. J. Med. Virol., 66: 388-393, 2002), make this enzyme moreprone to loss in activity, suggesting care is needed in fusing proteinsto the COOH terminus. The tk20rl fusion described in the current workhas a decreased TK activity that might be improved by using alonger/different spacer between the two genes or placing tk as thedownstream gene. On the other hand, this tk20rl fusion exhibits˜6-8-fold increase in RL activity in comparison to the rl alone that hasmade this fusion vector superior for bioluminescence imaging. However, atrue comparison of the activities of the fusion protein with thenonfused control proteins can be made only after measuring the K_(m) andV_(max) for each protein. Future studies will need to purify eachprotein and study the substrate kinetics in a detailed fashion to betterunderstand the effects of fusing the individual proteins on the proteinsability to act on substrate. Our results demonstrate that despitedecreased TK activity, it is possible to image the TK20RL fusion proteinnoninvasively and repetitively in living mice both by microPET and by anoptical CCD camera. Therefore, this fusion reporter gene has thepotential to translate approaches from small animal models topreclinical and clinical applications. We know of only one report in theliterature on measuring bioluminescence at the single cell level usingfl (Hooper, C. E., Ansorge, R. E., Browne, H. M., and Tomkins, P. CCDimaging of luciferase gene expression in single mammalian cells. J.Biolumin. Chemilumin., 5: 123-130, 1990) that required a highlysensitive CCD camera attached to a microscope. We are currentlyexploring the possibility to image the tk20rl fusion reporter at thesingle cell level using a similar setup. However, this approach mightnot be as useful as fluorescence approaches for cell imaging because ofthe relatively high light yield of fluorescence approaches as comparedwith bioluminescence approaches, and thus we are currently validating atriple fusion construct harboring a fluorescence (gfp or redfluorescence protein), a bioluminescence (fl or rl), and a PET (sr39tkor wild-type tk) reporter gene (Ray, P., Min, J. J., and Gambhir, S. S.Multimodality imaging of reporter gene expression in single cells andliving mice using a novel triple fusion vector. J. Nucl. Med., in press,2003). Although approaches have been validated to image fluorescencereporter gene expression in small living animals (Yang, M., Baranov, E.,Jiang, P., Sun, F. X., Li, X. M., Li, L. N., Hasegawa, S., Bouvet, M.,Al-Tuwaijri, M., Chishima, T., Shimada, H., Moossa, A. R., Penman, S.,and Hoffman, R. M. Whole-body optical imaging of green fluorescentprotein expressing tumors and metastases. Proc. Natl. Acad. Sci. USA,97: 1206-1211, 2000), bioluminescence reporter genes should exhibitseveral advantages for in vivo imaging in living animals. In contrast tofluorescence imaging, bioluminescence imaging is not limited by theautofluorescence properties of living cells, does not require anyexternal source of light for activation, and rather depends on thedelivery of specific substrates. Although there are reports of imagingfluorescence proteins in deep tissues of mice, these approaches oftenrequire special surgical procedures for exposing the animals. Incontrast, we can easily detect and quantitatively and reproduciblyevaluate the bioluminescence signal from various sites within the intactliving mouse as described in our previous reports (Wu, J. C., Inubushi,M., Sundaresan, G., Schelbert, H. R., and Gambhir, S. S. Optical imagingof cardiac reporter gene expression in living rats. Circulation, 105:1631-1634, 2002; Bhaumik, S. and Gambhir, S., supra, Proc. Natl. Acad.Sci. USA, 99: 377-382).

The fusion reporter gene described here may have some limitationsbecause of partial cleavage (−25-35%) of the fusion into its twocomponent proteins, which might result in a loss of sensitivity indifferent cell lines depending on the presence of specific proteases.However, the high correlation of TK and RL activities of the stablyexpressing cell lines in both in vitro and in vivo suggest that thisfusion will be useful in monitoring tumor growth and cell traffickingstudies where steady-state expression is expected. Future studies willneed to explore alternate spacers to minimize cleavage of the fusionprotein.

The higher sensitivity of optical imaging allows lower levels ofreporter gene expression and/or lower numbers of expressing cells to beimaged relative to the PET approach. The sensitivity differences cannotbe accounted for because of the reduced TK activity alone because thiswould only account for a 3-5-fold difference, and the number of cellsdetectable by optical imaging are several log-fold lower. We couldfollow reporter gene expression level using the fusion protein from avery early stage of s.c. implanted cells using the cooled CCD camera.Additional studies will be needed to better characterize the differencesin sensitivity at various depths within a mouse. However, the drawbackof bioluminescence imaging is this approach is not tomographic anddifficult to translate into humans. Presence of the PET reporter gene inthis fusion protein, on the other hand, is compatible with tomographictools for measuring reporter gene expression that could also be used inlarger subjects including humans. This fusion protein, therefore,provides a unique tool of validating different approaches quickly insmall animal models at a very low number of cells that can be rapidlytranslated to clinical use.

Future use of this fusion, including single cell imaging, should fosteradditional implementation of reporter genes directly from the cell toanimal to human level. This, in turn, should lead to acceleration ofmany areas of cancer research, including cell trafficking, tumortherapy, and gene therapy.

Example 2 Triple Fusion Reporter

Introduction: We have described the construction and validation of adouble fusion reporter vector (tk20rl) (Example 1) carrying a mutantHerpes Simplex Virus thymidine kinase (sr39tk) PET reporter gene and arenilla luciferase (rl) bioluminescence optical reporter gene in livingmice. In this Example, we describe the construction and testing ofseveral triple fusion reporter genes compatible with bioluminescence,fluorescence and positron emission tomography (PET) imaging.

A triple fusion reporter vector harboring a bioluminescence syntheticRenilla luciferase (hrl) reporter gene, a reporter gene encoding themonomeric red fluorescence protein (mrfp1), and a mutant herpes simplexvirus type 1 sr39 thymidine kinase [HSV1-truncated sr39tk (ttk); a PETreporter gene] was found to preserve the most activity for each proteincomponent and was therefore investigated in detail. After validating theactivities of all three proteins encoded by the fusion gene in cellculture, we imaged living mice bearing 293T cells transiently expressingthe hrl-mrfp-ttk vector by microPET and using a highly sensitive cooledcharge-coupled device camera compatible with both bioluminescence andfluorescence imaging. A lentiviral vector carrying the triple fusionreporter gene was constructed and used to isolate stable expressers byfluorescence-activated cell sorting. These stable 293T cells werefurther used to show good correlation (R² ˜0.74-0.85) of signal fromeach component by imaging tumor xenografts in living mice with all threemodalities. Furthermore, metastases of a human melanoma cell line(A375M) stably expressing the triple fusion were imaged by microPET andoptical technologies over a 40-50-day time period in living mice.Imaging of reporter gene expression from single cells to living animalswith the help of a single tri-fusion reporter gene will have thepotential to accelerate translational cancer research.

Methods:

Chemicals. [8-³H] Penciclovir and ¹⁴C-labeled2′-fluoro-5-fluoro-1-β-D-arbinofuranosyluracil were obtained fromMoravek Biochemicals (Brea, Calif.). 9-(4-[¹⁸F]Fluoro-3-hydroxymethylbutyl) guanine (FHBG) was also synthesized atUniversity of California Los Angeles as detailed previously (Yaghoubi,S., Barrio, J. R., Dahlbom, M., Iyer, M., Namavari, M., Satyamurthy, N.,Goldman, R., Herschman, H. R., Phelps, M. E., and Gambhir, S. S. Humanpharmacokinetic and dosimetry studies of [¹⁸F] FHBG: a reporter probefor imaging herpes simplex virus type-1 thymidine kinase reporter geneexpression. J. Nucl. Med., 42: 1225-1234, 2001). Coelenterazine waspurchased from Biotium, Inc. (Hayward, Calif.). The polyclonal anti-TKantibody was a kind gift of Dr. M. Black (Washington State University,Pullman, Wash.), and the monoclonal anti-Renilla luciferase protein (RL)was purchased from Chemicon International (Temecula, Calif.).

Construction of hrl-mrfp-ttk and Other Fusion Genes. PCR amplificationand standard cloning techniques were used to insert the hrl and mrfpgenes from plasmid pcDNA 3.1-CMV-hrl (Promega, Madison, Wis.) andpcDNA3.1-CMV-mrfp1 in frame with the ttk gene into thepcDNA3.1-sr39-truncated tk (a kind gift of Dr. D. Kaufman; University ofCalifornia, Los Angeles, Calif.). The CMV-wtk vector was obtained fromDr. M. Black and modified to truncated wtk (wttk) by deleting first 135bp through PCR and cloned in pcDNA3.1 backbone to generate CMV-wttkplasmid. CMV-fl and CMV-egfp were purchased from Promega and BDSciences-Clontech (Palo Alto, Calif.) respectively. For PCRamplifications, different 5′ and 3′ end primers were used to generatethe fusion vectors. Standard cloning techniques were used to generatethe lentiviral (CS-hrl-mrfp-ttk) vector as performed previously in ourlaboratory (De, A., Lewis, X. H., and Gambhir, S. S. Noninvasive imagingof lentiviral-mediated reporter gene expression in living mice. Mol.Ther., 7: 681-691, 2003).

Cell Lines and Transient Transfection Procedures. Neuro 2a (N2a)neuronal cell lines (a gift from Dr. Vincent Mauro; Scripps ResearchInstitute, La Jolla, Calif.), 293T human embryonic kidney cells(American Type Culture Collection, Manassas, Va.), and A375M humanmelanoma cells (a gift from Dr. M. Kolodny; University of California,Los Angeles, Calif.) were used. The N2a and A375M cells were cultured inhigh-glucose DMEM supplemented with 10% fetal bovine serum and 1%penicillin (100 μg/ml) and streptomycin (292 μg/ml), and 293T cells weregrown in MEM supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin solution. All transient transfections werecarried out using the Superfect transfection reagent (Qiagen, Valencia,Calif.) following the protocol recommended by the manufacturer.

tTK, hRL, and β-Galactosidase (β-Gal) Activity. TK enzyme activityassays were performed as described previously (Gambhir, S. S., Bauer,E., Black, M. E., Liang, Q., Kokoris, M. S., Barrio, J. R., Iyer, M.,Namavari, M., Phelps, M. E., and Herschman, H. R. A mutant herpessimplex virus type 1 thymidine kinase reporter gene shows improvedsensitivity for imaging reporter gene expression with positron emissiontomography. Proc. Natl. Acad. Sci. USA, 97: 2785-2790, 2000), and β-Galand Renilla or firefly luciferase assays were done using the (β-Galenzyme assay system and Dual-Luciferase Reporter Assay System fromPromega, respectively. Each of the luciferase reactions was measured ina TD 20/20 luminometer (Turner Designs, Sunnyvale, Calif.) for a periodof 10 s.

Western Blot Analysis. The levels of tTK and hRL were evaluated byWestern blotting with a rabbit polyclonal anti-TK antiserum and a mousemonoclonal anti-Renilla antibody using cell lysates prepared from 293Tcells transfected with CMV-hrl-mrfp-ttk, CMV-ttk, and CMV-hrl plasmids.

Lentiviral Production. Lentivirus was developed and used to infect 293Tand A375M cells as described previously (De, A., Lewis, X. H., andGambhir, S. S. Noninvasive imaging of lentiviral-mediated reporter geneexpression in living mice. Mol. Ther., 7: 681-691, 2003).

Fluorescence Microscopy, CCD Imaging, and FACS. Expression of mRFP1 wasobserved under a Zeiss Axiovert 200M fluorescence microscope (Carl ZeissMicroimaging Inc., Thornwood, N.Y.) with DsRed filter setting 546 nm;λ_(ex), 605 nm) and analyzed with MetaMorph software (University ImagingCorp., Downingtown, Pa.). For quantification of the expression level ofmrfp1 present in the CMV-hrl-mrfp-ttk and CMV-mrfp1, 1×10⁴ and 1×10⁵ of293T, A375M, or N2a cells expressing the vectors were seeded inblack-bottomem clear 96-well plates and imaged in the Xenogen IVISoptical imaging system (Xenogen Corp., Alameda, Calif.) with anexcitation filter at 500-550 nm and an emission filter at 575-650 nm.Regions of interest (ROIs) were drawn over the cell area and quantifiedby using Living Image Software version 2.20. For FACS, 1×10⁶ ofCS-hrl-mrfp-ttk infected 293T and A375M cells were sorted by using aBecton Dickinson FACSvantage SE cell sorter.

MicroPET Imaging of Mice. Animal care and euthanasia were performed withthe approval of the University of California Animal Research Committee.Male 12-14-week-old nude mice (nu/nu) received s.c. injection with˜10×10⁶ 293T cells transiently expressing the CMV-hrl-mrfp-ttk fusion,CMV-ttk, CMV-hrl, and CMV-mrfp1 on the ventral side, and mice (n=4) werescanned the next day using a microPET as described previously (Gambhir,S. S., supra, Proc. Natl. Acad. Sci. USA, 97: 2785-2790). Additionally,10×10⁶ of each of four differentially expressing clones of 293T cellsstably expressing hrl-mrfp-ttk gene were implanted in three mice andscanned in the microPET ˜24 h later. The microPET images werereconstructed by using three-dimensional filtered back projection and aniterative maximum a posteriori algorithm (Qi, J. Y., Leahy, R. M.,Cherry, S. R., Chatziioannou, A., and Farquhar, T. H. High-resolution 3DBayesian image reconstruction using the microPET small-animal scanner.Phys. Med. Biol., 43: 1001-1013, 1998). ROIs were drawn over the tumorarea. The ROI counts were converted to percentage of injected dose/g(ID/g) using filtered back projection as described previously (Gambhir,S. S., supra, Proc. Natl. Acad. Sci. USA, 97: 2785-2790), and imagesshown were reconstructed with maximum a posteriori algorithm.

Bioluminescence and Fluorescence Imaging of mRFP1 and RL Expression inLiving Mice. For in vivo fluorescence imaging, mice implanted with thecells described above were anesthetized, and each mouse was placed in alight tight chamber equipped with a halogen light source, and whole bodyimage was acquired for 1 s using the Xenogen IVIS optical imaging systemwith an excitation filter at 500-550 nm and an emission filter at575-650 nm. ROIs were drawn over implanted cell area and quantified byusing Living Image Software version 2.20. For bioluminescence imaging,each mouse next received injection with 10 μl (2 μg/μl dissolved inmethanol) of coelenterazine diluted in 90 μl of PBS (pH 7) via tailvein. Each animal was then placed supine in the same light tightchamber, and whole body images were obtained and quantified as describedpreviously (Bhaumik, S., and Gambhir, S. S. Optical imaging of Renillaluciferase reporter gene expression in living mice. Proc. Natl. Acad.Sci. USA, 99: 377-382, 2002). Both bioluminescence and fluorescencesignals were recorded as maximum [photons/second/centimeter²/steradian(photons/s/cm²/sr)].

Multimodality Imaging of Cancer Metastasis in Living Mice Using a HumanMelanoma Cell Line (A375M) Stably Expressing the Triple Fusion Vector.Three 8-week-old Beige severe combined immunodeficient mice receivedinjection with 7×10⁵ A375M cells stably expressing the hrl-mrfp-ttk genevia tail vein and were imaged repeatedly with fluorescence,bioluminescence, and microPET. At day 40, the mice were first imagedwith microPET and bioluminescence (as described above) and thensacrificed and imaged; the chest was cut open with Illuminatool Tunablelighting system using the 540 nm excitation filter and RFP viewing glass(Lightools Research). Fluorescence imaging and light photograph of micewere digitally captured with a Nikon camera for 2 s.

Results.

A Multimodality Fusion Vector Harboring the hrl Gene (Bioluminescence),Gene Encoding for mRP1 (Fluorescence), and a Mutant Truncated HSV1-sr39Thymidine Kinase (PET) Reporter Gene Maintains hRL, mRFP1, and tTKActivity in Several Cell Lines. We first constructed a fusion genevector (hrl-ttk) carrying hrl (hrl, gene; hRL, enzyme) and ttk (ttk,gene; tTK, enzyme) reporter genes separated by a 22-aa-long spacer(LENSHASAGYQACGTAGPGSTG) (SEQ ID NO: 8) and then inserted thePCR-amplified mrfp1 gene fragment in the middle of the spacer (at theposition of Cys-Gly) to generate a hrl-mrfp-ttk triple fusion reportergene. The PCR-amplified hrl gene fragments from pCMV-hrl vector wereinserted in frame with ttk gene (the first 45 aa of sr39tk gene weretruncated to delete the nuclear localization signal of tk gene) clonedin pcDNA3.1+ separated by the above-mentioned spacer under the controlof a CMV promoter. The resultant vector was then digested with HindIIIand SacII and ligated in frame to PCR-amplified andHindIII/SacII-digested mrfp1 fragments (without stop codon) from pRSETBvector to generate the hrl-mrfp-ttk fusion vector. The order of thethree different reporter genes and spacers in this triple fusion vectoris as follows: hrl-spacer (LENSHASAGYQAST) (SEQ ID NO: 9)-mrfp-spacer(TAGPGSAT) (SEQ ID NO: 10)-ttk gene. The final vector was fully verifiedby sequencing.

Plasmid DNA prepared from four to five clones of CMV-hrl-mrfpttk triplefusion were transiently transfected in 293T cells, and the cells werefirst observed in a fluorescence microscope for mrfp1 activity andfurther assayed for hRL and tTK activity. The plasmid clone exhibitingthe highest mRFP1, hRL, and tTK activities was selected for additionalstudies. To extend our study to different variants ofbioluminescence/fluorescence/PET reporter genes, we also generatedseveral functionally active multimodality reporter fusion vectors, i.e.,fl-mrfp-ttk [by replacing hrl with firefly luciferase (fl)],flllzrl-egfp/rfp-ttk (mrfp1 is replaced with egfp or tetrameric rfpknown as DsRed2), and fl/hrl-rfp-wttk [by replacing the truncatedHSVI-sr39tk (ttk) with wild-type HSV1-truncated thymidine kinase(wttk)]. The nature and the order of the spacers for all theseconstructs were equivalent to CMV-hrl-mrfp-ttk vector described earlier.The ttk, wttk, fl, hrl, rfp, gfp, and mrfp1 genes were also cloned inpcDNA3.1+ backbone to generate positive control plasmids to directlycompare the results of each fusion. All these fusion vectors werefunctionally active with respect to each individual protein, however thelevel of activity varied for each construct (Table 1). Overall, thehrl-mrfp-ttk fusion construct showed the highest activity for all threeof the component proteins in comparison with other vectors and thus wasfurther studied for multimodality imaging.

TABLE 1 Fluorescent, bioluminescent and PET^(a) reporter geneexpressions exhibited by different triple fusion constructs incomparison to the respective positive controls assayed from transientlytransfected 293T cells (TTK and hRL activites are normalized withco-transfected β-Gal activity RFP/eGFP/mRFP (fluorescence % TK % wTK %hRL % FL activity by Constructs activity activity activity activitymicroscopy) hrl-rfp-ttk 39.7 27.8 Medium fl-rfp-ttk 29.6 22.1 Lowhrl-egfp-ttk 50 33 High fl-egfp-ttk 43 20 High hrl-mrfp-ttk 149 54 Highfl-mrfp-ttk 100 53.6 Medium hrl-rfp-wttk 76 44.7 Medium fl-rfp-wttk 61.662.6 Low ttk 100 wttk 100 hrl 100 fl 100 mrfp1 Very high ^(a)PET,positron emission tomography; eGFP, enhanced green fluoresence protein;β-Gal, β-galatosidase,

To compare the levels of reporter gene expression of each of thecomponents of the CMV-hrl-mrfp-ttk plasmid, three different cell lines[293T, N2a, and A375M (FIG. 5, A-C) were transiently transfected withthe triple fusion plasmid along with proper positive controls(pcDNA3.1-ttk, pcDNA3.1-mrfp1, pcDNA3.1-hrl) and negative controls(mock-transfected control cells). Each cell line was also cotransfectedwith the CMV-β-gal reporter gene to normalize for transfectionefficiency. After 24 h, the expression of mrfp1 was observed in theinverted fluorescence microscope, and then activity of the other threereporter genes was assayed from the same cell lysates, and tTK and hRLactivities were normalized to β-Gal activity. In all three of thedifferent cell lines, CMVhrl-mrfp-ttk showed equal or slightly highertTK activity (not statistically significant) compared with the positivecontrol (pcDNA3.1-ttk) but had a lower hRL activity [33% (A375M), 27.4%(N2a), and 54% (293T)] compared with that of the positive controlpcDNA3.1-hrl plasmid (P<0.05). The expression level of mrfp1 of thistriple fusion vector in all of the cell lines was 60-70% of the positivecontrol pcDNA3.1-mrfp1 vector, as determined by the fluorescence signalusing the CCD camera.

Western Blot Analysis of Extracts from Cells Transiently Transfectedwith hrl-mrfp-ttk Probed with Anti-TK and Anti-RL Antibodies Reveals thePresence of a 100-kDa Fragment, the Predicted Size of the ProteinEncoded by the hrl-mrfp-ttk Fusion. To investigate hrl-mrfp-ttk fusionreporter gene expression at the protein level, cell lysates fromhrl-mrfp-ttk-, ttk-, and hrl-transfected 293T cells were resolved by 10%SDS-PAGE and analyzed on Western blots by using antibodies specific forTK and RL (FIG. 6A). The predominant band in the triple fusion samplerecognized by anti-TK (FIG. 6A.1) and anti-RL (FIG. 6.A.2) antibodies isabout 100 kDa, the expected size of the hRL-mRFP-tTK fusion protein. ThetTK and the hRL proteins were recognized at about 36 kDa band by theirspecific antibodies. Similar results were also obtained from N2a andA375M cell extracts (data not shown).

A Lentiviral Vector Carrying the hrl-mrfp-ttk Triple Fusion Is Able toStably Transfect 293T and A375M Cells, and the Positive Expressers CanBe Sorted by FACS Analysis. One potential use of a lentiviral vector isto deliver the genes of interest to any type of dividing or nondividingcell lines or target tissues in living animals. Therefore, thefull-length fusion gene cassette was cloned in the NheI and XhoI site ofa second-generation lentiviral vector (De, A., Lewis, X. H., andGambhir, S. S. supra, Mol. Ther., 7: 681-691), and viruses carrying thetriple fusion reporter gene were used to transduce 293T cells. Fivemillion 293T cells infected with lentivirus were sorted by FACS using a585±42 nm filter setting, and 33% positive expressers were collected(FIG. 6B.1). The sorted and unsorted cells were then assayed for tTK andhRL expression (FIG. 6B.2) and clearly show a significant (P<0.05) gainin expression in the sorted cell population. Similarly, the A375M cellswere transduced with the lentivirus carrying the triple fusion reportergene, and positive expressers were selected by two rounds of FACSsorting and verified to have significant hRL and tTK activities (datanot shown).

293T Cells Transiently Expressing the hrl-mrfp-ttk Fusion Reporter GeneCan Be Imaged in Living Mice with the MicroPET and Optical Cooled CCDImaging Systems. In order to test the fusion reporter vector for itsefficacy in simultaneous imaging of reporter gene expressionquantitatively and repeatedly in living subjects using differentmodalities, we injected 10×10⁶ 293T cells transiently transfected witheither CMV-hrl-mrfp-ttk, CMVttk, CMV-hrl, or CMV-mrfp1 vectors s.c. atfour different sites on the ventral sides of four 12-14-week-old nu/nunude mice. The mice were first scanned using the cooled CCD camera forfluorescence followed by a bioluminescence scan after injection of 20 μgof coelenterazine via tail vein. Fluorescence imaging of these micereveals that the cells expressing the hrl-mrfp-ttk fusion (FIG. 7A.1, a)show lower fluorescence [˜11.03±6.03×10⁸ maximum (p/s/cm²/sr)] incomparison with the cells expressing mrfp1 vector (FIG. 7A.1, c) alone[˜65.8±32.29×10⁸ maximum (p/s/cm²/sr); FIG. 7A.1]. No significant signalis observed from the other two sites of implantation carrying theCMV-ttk (FIG. 7A.1, b)- and CMV-hrl (FIG. 7A.1, d)-expressing cells. Abioluminescence scan of the mice shows a signal of 5.8±3.7×10⁶ maximum(p/s/cm²/sr) from the cells expressing the fusion reporter gene (FIG.7A.2, a) and about 7.37±4×10⁶ maximum (p/s/cm²/sr) from theCMV-hrl-expressing cells (FIG. 7A.2, d). Similar to fluorescenceimaging, the other two sites carrying CMV-mrfp1 (FIG. 7A.2, c)- andCMV-ttk (FIG. 7A.2, b)-expressing cells did not show any significantbioluminescence signal. Because the FHBG mass used for PET imaging is1000-fold lower (due to the presence of radioactive isotope) than thecoelenterazine mass used for bioluminescence imaging, PET imaging is notas sensitive as bioluminescence at superficial depths. Furthermore, PETimaging benefits from well-vascularized tissues with relatively highlevels of reporter gene expression. We therefore implanted the cellsexpressing the fusion gene and ttk gene in the right and left axillaryregion of the mice. We quantified the signal from each of the sitesexpressing the CMV-hrl-mrfp-ttk and CMV-ttk directly from the microPETimages to determine the percentage of ID/g tumor for FHBG. Thispercentage of ID/g is a measure of the amount of tracer accumulated in agiven tissue site normalized to the injected amount and to the mass ofthe tissue examined. The mean percentage of ID/g for FHBG accumulationin the CMV-hrlmrfp-ttk-expressing cells (0.303±0.09; FIG. 7A.3, a andFIG. 7A.4, a) did not differ significantly from that of theCMV-ttk-expressing cells (0.313±0.09; FIG. 7A.3, b and FIG. 7A.4, b) forthe four mice (FIG. 7A.3 and 7A.4). Preservation of a high level of tTKactivity and moderate levels of hRL and mRFP1 activities by thistri-fusion vector thus allows simultaneous imaging of transientexpression of all three of the reporter genes in living mice with allthree of the imaging techniques. Repetitive imaging of the same mouseover a 10-day period produced signals for all three of the reportergenes that increased with time as the tumor burden increased (data notshown).

The lentivirus-infected 293T cells stably expressing the hrl-mrfp-ttkfusion gene described in the previous section were diluted to singlecells, and four clones with differential expression of all three geneswere selected. In cell culture, these four clones exhibited goodcorrelation between tTK and hRL (R²=0.96), hRL and mRFP1 (R²=0.94), andtTK and mrfp1 (R²=0.86) activities (data not shown). For quantificationof mrfp1 activity, 3.5×10⁵ cells of each clone and 293T cells wereseeded in black, clear-bottomed 96-well plates in triplicate and imagedin the CCD camera with DsRed2 excitation and emission filter options.ROIs were drawn on each well, and fluorescence was measuredquantitatively for each group. The basal fluorescence exhibited by 293Tcells was subtracted from the fluorescence of each well of each clone,and the mean of the three wells of each clone was taken as absolutefluorescence activity, expressed as maximum (p/s/cm²/sr) for each clone.tTK and hRL assays were performed as described earlier. To measure thecorrelation between the expression of the three reporter genes acrossthree different imaging modalities, 10×10⁶ cells of each clone wereimplanted on the two axillary regions on the ventral side of three nudemice (two clones in each mouse), and mice were imaged for fluorescence,bioluminescence, and microPET on the same day as described above. Thefluorescence and bioluminescence signals and the percentage of ID/gvalues for FHBG of the hrl-mrfp-ttk expressing clones across the threemice were all well correlated: R²=0.85 (hRL:tTK; FIG. 7B.1); R²=0.74(hRL:mRFP1; FIG. 7.B.2); and R²=0.74 (tTK:mrfp1; FIG. 7B.3).

Metastasis of A375M Human Melanoma Cells Expressing the hrl-mrtp-ttkReporter Gene Can Be Imaged by MicroPET and an Optical CCD Camera inLiving Mice. To apply the tri-fusion strategy to a relevant preclinicalcancer study, we used a melanoma metastatic model. A375M human melanomacells are known to metastasize to other organs once injected in theanimal i.v. and form pulmonary and brain metastases with some rareoccurrence of bone metastases. A375M cells (7×10⁵) stably expressing thehrl-mrfp-ttk reporter gene were injected in three 8-week-old Beigesevere combined immunodeficient mice via tail vein. On the first day ofcell injection, bioluminescence signal was detectable from the lungs(the primary route of cell migration; FIG. 8A), but not from microPETimages (data not shown). The mice were then subsequently imaged overtime every 6-7 days for a period of 40-50 days. At day 40, moderatemicroPET signal (˜0.35% ID/g) from the lungs and strong signal (˜0.78%ID/g) from the chest region are detected from one of the three mice(FIG. 8C). A corresponding bioluminescence signal (2×10⁵ p/s/cm²/sr) isdetected from the lungs of the same mouse on the same day (FIG. 8B). Therelatively high PET signal from the chest region (FIG. 8C) is notevident with bioluminescence imaging (FIG. 8C), likely due to relativelypoor penetration of light produced by Renilla luciferase from greaterdepths. A faint bioluminescence signal (5×10³ p/s/cm²/sr) was also seenfrom the pelvic region of the mouse that was undetectable in microPET,likely due to hindrance by the nonspecific signal due to FHBG tracerclearance from the kidneys and gastrointestinal tract. In vivofluorescence imaging of metastases did not produce good images due tosignificant autofluorescence caused by the presence of hair. However,when the mouse was sacrificed, and internal organs were exposed, severalsmall metastatic tumors were found with fluorescence (FIG. 8E). Amongthe other two mice, one showed bioluminescence signal in the abdominalregion at day 48; however we could not detect specific microPET signal,likely due to the presence of moderate levels of nonspecific signalresulting from the clearance of FHBG through kidneys and thegastrointestinal tract.

Discussion.

We describe herein the construction of several novel triple fusionreporter genes including one harboring a bioluminescence reporter gene(synthetic Renilla luciferase), a fluorescence reporter gene (monomericred fluorescence protein), and a PET reporter gene (truncated version ofHSV1-sr39 thymidine kinase) and validate its application in living cells(cell microscopy and FACS) and in living mice using three differentsmall animal imaging technologies (in vivo fluorescence, in vivobioluminescence, and microPET).

Use of bifusion reporter genes for molecular imaging has been validatedpreviously by us (see Example 1; see also, Ray, P., Wu, A., and Gambhir,S. Optical bioluminescence and positron emission tomography imaging of anovel fusion reporter gene in tumor xenografts of living mice. CancerRes., 63: 1160-1165, 2003) and by other investigators (Jacobs, A.,Dubrovin, M., Hewett, J., Sena-Esteves, M., Tan, C. W., Slack, M.,Sadelain, M., Breakefield, X. O., and Tjuvajev, J. G. Functionalcoexpression of HSV-1 thymidine kinase and green fluorescent protein:implications for noninvasive imaging of transgene expression. Neoplasia,1: 154-161, 1999; Wang, Y., Yu, Y., Shabahang, S., Wang, G., and Szalay,A. Renilla luciferase-Aequorea GFP (Ruc-GFP) fusion protein, a noveldual reporter for real-time imaging of gene expression in cell culturesand in live animals. Mol. Genet. Genomics, 268: 160-168, 2002). Theseprevious approaches have been limited by the inability to image singlecells (Ray) or to take advantage of the low background signal withbioluminescence (Jacobs) or the advantage of tomographic imaging withPET (Wang). However, the ability to have a fluorescence,bioluminescence, and PET signal provides the full spectrum of coverageneeded for many reporter gene applications. One can use this tri-fusionreporter gene to sort cells and to image in small living animals usingeither fluorescence or bioluminescence and in larger subjects, includinghumans, using PET. The ability to move between imaging technologieswithout having to use a different reporter gene for each applicationwill greatly simplify various biological models including transgenics,cell trafficking, anticancer pharmaceutical research, and gene therapy.

Although our previous bi-fusion reporter construct (tk₂₀rl) showedwell-correlated expression of PET and bioluminescence imagingmodalities, it was somewhat limited by decreased TK activity and wassusceptible to enzymatic cleavage into its component proteins. Bychanging the orientation of the fusion partner in the current vector, wecould gain a significant amount of TK activity, indicating that theCOOH-terminal end of thymidine kinase protein may be crucial forensuring TK enzyme activity. In contrast, the hRL activity of thecurrent construct showed a decrease in enzyme activity as opposed to ourprevious vector, which showed a gain in RL activity. However, this newsynthetic version of Renilla luciferase (hRL) is 40-50-fold more activethan original Renilla luciferase (Bhaumik, S., Lewis, X. Z., andGambhir, S. S. Optical imaging of synthetic Renilla luciferase reportergene expression in living mice. J. Biomed. Optics, in press, 2004), andtherefore a drop in RL activity did not affect the efficacy of thisvector significantly. Moreover, the tk gene in this triple fusion vectorhas a deletion of the first 135 bp that contains a nuclear localizationsignal and a cryptic testis-specific transcriptional start point(Degreve, B., Johansson, M., Declercq, E., Karlsson, A., and Balzarini,J. Differential intracellular compartmentalization of herpetic thymidinekinases (TKS) in TK genetransfected tumor cells. Molecularcharacterization of the nuclear localization signal of the herpessimplex virus type 1 TK. J. Virol., 72: 9535-9543, 1998; Cohen, J. L.,Boyer, O., Salomon, B., Onclercq, R., Depetris, D., Lejeune, L.,Dubus-Bonnet, V., Bruel, S., Charlotte, F., and Mattei, M. G. Fertilehomozygous transgenic mice expressing a functional truncated herpessimplex thymidine kinase TK gene. Transgenic Res., 7: 321-330, 1998).Thus, this deletion leads to more cytoplasmic localization of TK enzyme,likely resulting in more TK activity (Luker, G. D., Sharma, V., Pica, C.M., Dahlheimer, J. L., Li, W., Ochesky, J., Ryan, C. E., Piwnica-Worms,H., and Piwnica-Worms, D. Noninvasive imaging of proteinproteininteractions in living animals. Proc. Natl. Acad. Sci. USA, 99:6961-6966, 2002) due to the availability of greater amount of substrate(BBG). This deletion mutant will also likely overcome the problem ofmale sterility in transgenic mice carrying the thymidine kinase gene dueto production of a shorter transcript in testis from a cryptictranscriptional initiation site present in the first 135 bp of the gene.Another added advantage of this vector over our previous one and othervectors reported in the literature is that it can retain its integrityas a fusion protein when expressed, so that signal from each componentof the tri-fusion protein will not be susceptible to problems related tocleavage. The absence of cleavage of the triple fusion vector is likelydue to change of certain amino acids (Cys-Gly to Ser-Thr) present in thespacer in contrast to the previously reported 20-aa spacer of the tk20rlvector.

In the process of building a better multimodality vector, we constructedseveral other fusion vectors (see Table 1). Most of these vectors hadlower tTK, luciferase, and RFP activity, probably due to the inherentnature of RFP (DsRed2) of forming obligate tetramer for propermaturation of the fluorophores present in the fusion genes. Thetetrameric nature of RFP present in hrl/fl-rfp-ttk fusions might imposestructural and functional constrains on the other partner proteinsresulting in decreased TK and luciferase activity. Our hrl/fl-egfpttkvectors did show a better TK and luciferase activity than hrl/fl-rfp-ttkvectors, due to the monomeric nature of eGFP proteins, but did not showbetter activity than the hrl/fl-mrfp-ttk fusion vectors. Moreover, theexcitation and emission spectra of GFP (λ_(ex) 489 nm; λ_(ex), 508 nm)is not as favorable for fluorescence imaging in living subjects ascompared with RFP and mRFP because of the better penetration of red andnear-infrared light in tissues. We also consistently observed a drop intTK and RFP activity of the triple fusions with firefly luciferase incomparison with the fusions bearing Renilla luciferase. Fusion reportervectors bearing truncated wild-type thymidine kinase also preserved abetter wild-type thymidine kinase and luciferase activity (with bothfirefly and Renilla), and these vectors should be useful in the futurewhen using other substrates (e.g.,2′-fluoro-5-fluoro-1-β-D-arbinofuranosyluracil/2′-deoxy-2′-fluoro-5-fluoro-l-p-D-arbinofuranosyluracil)that are more sensitive when used with wild-type thymidine kinase. It islikely that one tri-fusion will not serve the needs for allapplications, and investigators will need to choose from a library oftri-fusions for a given application.

One of the potential uses of the multimodality reporter vectors in genetherapy is to target any type of cell line or tissues and then followgene expression using a multimodality approach. Viral vectors,especially the lentiviral ones, are among the most standardized andwidely used vectors to deliver any gene of interest to target tissues oran organism and to isolate cells, particularly nondividing cells stablyexpressing the gene. Recently, a bicistronic lentiviral vector carryingtk and fl reporter genes has been successfully used for PET andbioluminescence imaging in our laboratory (De, A:, Lewis, X. H., andGambhir, S. S. Noninvasive imaging of lentiviral-mediated reporter geneexpression in living mice. Mol. Ther., 7: 681-691, 2003). The newlentiviral construct carrying the triple fusion gene described hereinhas been used successfully with FACS analysis to isolate lentiviralinfected 293T and A375M cells stably expressing the triple fusionreporter. This lentiviral construct should have tremendous potential inwide variety of research applications. Our data with the A375Mmetastatic melanoma model further confirm the usefulness of thislentiviral vector carrying triple fusion reporter gene to followprogression of cancer metastases by molecular imaging. Extensions ofthis study with drug treatment are currently in progress (De, A.,Collison, E., Kolodney, M., and Gambhir, S. Lentiviral reporter genedelivery as a novel way of studying therapeutic effects on cancermetastasis by noninvasive imaging. Mol. Ther., 7: S137, 2003).

Use of light is probably the oldest method of analyzing tissues inbiomedical science. The various optical imaging approaches includingfluorescence microscopy (at the cellular level), diffuse opticaltomography, and intravital microscopy (for deeper structures at theorganism level) are commonly used (Boas, D. A., Brooks, D. H., Miller,E. L., DiMarzio, C. A., Kilmer, M., Gaudette, R. J., and Zhang, Q.Imaging the body with diffuse optical tomography. IEEE Signal ProcessingMagazine, 18: 57-75, 2001; Jain, R. K., Munn, L. L., and Fukumura, D.Dissecting tumour pathophysiology using intravital microscopy. Nat. Rev.Cancer, 2: 266-276, 2002). However, intrinsic absorption and scatteringof light through the tissues and autofluorescence properties ofbiological molecules (e.g., tryptophan, collagen, elastin, nicotinamideadenine dinucleotide, hemoglobin, oxyhemoglobin, and so forth) imposecertain restrictions for using fluorescence as an imaging tool in smallliving subjects. However, both light attenuation and autofluorescencedecline as wavelength increases, especially in the red to near-infraredregion (>600 nm). A fluorescence protein/fluorochrome with excitationand emission toward red (560 nm onward) has better penetrability throughthe tissues than that with excitation and emission in the blue or greenregion. Moreover, hemoglobin and water, which are responsible for thehighest absorption of light among all other biological molecules, havetheir lowest coefficient of absorption in the red and near-infraredregion.

Therefore, in vivo fluorescence imaging is more suitable in the red andnear-infrared region than in the green or yellow region. Optical imagingin living subjects at the near-infrared region (650-900 nm) hastherefore been used extensively by applying different fluorochromes thatemit light at near-infrared region spectra and in combination with otherimaging modalities (Ntziachristos, V., Bremer, C., and Weissleder, R.Fluorescence imaging with near-infrared light: new technologicaladvances that enable in vivo molecular imaging. Eur. Radiol., 13:195-208, 2002; Zaheer, A., Lenkinski, R. E., Mahmood, A., Jones, A. G.,Cantley, L. C., and Frangioni, J. V. In vivo near-infrared fluorescenceimaging of osteoblastic activity. Nat. Biotechnol., 19: 1148-1154,2001). However, synthesis and attachment of these fluorochromes toproteins/copolymers require complex chemical procedures and are moredifficult to generalize. Also, these strategies are not directlyapplicable to genetically encoded reporters. The mRFP1 protein used inthis study has an excitation and emission range in the far-red region(584-607 nm) and thus is one of the better reporter gene choices forfluorescence imaging in living subjects (Campbell, R. E., Tour, O.,Palmer, A. E., Steinbach, P. A., Geoffrey, S., Baird, G. S., David, A.Z., and Tsien, R. Y. A monomeric red fluorescent protein. Proc. Natl.Acad. Sci. USA, 99: 7877-7882, 2002). The monomeric nature of thisprotein also confers a better functional preservation as fusion partneras compared with RFP or DsRed2 (tetramer) and HcRed (dimer). However, westill observed a significant amount of autofluorescence from the mouserelative to bioluminescence, which is not limited by autofluorescence,and therefore bioluminescence produced a better signal: background ratioin living animals. However, bioluminescence imaging of gene expressionin a single cell is not easily possible due to generation of relativelylow amounts of light. Therefore, the current results would support usingthe fluorescence component for cell imaging/sorting with limited in vivoimaging, bioluminescence for small animal imaging even with a very fewnumber of cells, and PET for tomographically imaging living subjectsincluding larger animals and humans.

Our cancer metastatic model shows that metastases can be imaged bymicroPET and bioluminescence in living mice using this triple fusionreporter gene, with certain limitations for each technique. Thebioluminescence signal from Renilla luciferase is not detectable frommetastases present at greater depths but is easily detectable fromsuperficial metastases from any region of the body. On the other hand,microPET reveals metastases from deep inside the body, but signals frommetastases in the abdomen/pelvis are somewhat obscured by thenonspecific signal in the gastrointestinal tract and urinary collectingsystem due to tracer clearance. Finally, autofluorescing properties ofbiological molecules limit detection of metastases by in vivofluorescence imaging in living animals.

However, metastases can be easily visualized in sacrificed animals withexposed tissues in situ using whole body fluorescence imaging. It islikely that the fluorescence signal to background can be improved byusing an excitation source under the animal and imaging with a cameraabove the animal to help minimize autofluorescence. The bioluminescencesignal can also be markedly improved by injecting higher doses ofsubstrate (coelenterazine), as we have demonstrated recently (Bhaumik,S., Lewis, X. Z., and Gambhir, S. S. Optical imaging of syntheticRenilla luciferase reporter gene expression in living mice. J. Biomed.Optics, in press, 2004). As red-shifted bioluminescent reporters withhigh substrate utilization capacity are developed, this will also likelyaid in helping to use bioluminescence-based reporter fusions. Inaddition, the microPET signal can be improved by using tracers withlonger half-lives (e.g., ¹²⁴1_labeled2′-fluoro-5-fluoro-1-β-D-arbinofuranosyluracil) to allow the backgroundsignal from the gastrointestinal tract and renal collecting system to bereduced by waiting longer after tracer injection before imaging animals.With continued refinement in reporter genes, substrates for reporterproteins, and physical instrumentation, it is likely that higher spatialresolution imaging with greater sensitivity for detecting smallernumbers of cells will eventually be possible.

Additional studies quantitatively comparing fluorescence,bioluminescence, and PET in small living animals should also help tobetter define the potential roles of each modality in specificapplications. Cancer research, including imaging of preclinical modelsof tumors and metastases, immune cell trafficking, transgenic models,gene therapy, and monitoring therapy in general should all benefit fromthe strategies described herein.

While this invention has been described in detail with reference to acertain preferred embodiments, it should be appreciated that the presentinvention is not limited to those precise embodiments. Rather, in viewof the present disclosure which describes the current best mode forpracticing the invention, many modifications and variations wouldpresent themselves to those of skill in the art without departing fromthe scope and spirit of this invention.

In particular, it is to be understood that this invention is directed toany fusion vector coupling any fluorescent protein (including, but notlimited to, green, yellow and red fluorescent protein) and anybioluminescent protein (including, but not limited to, RenillaLuciferase and Firefly Luciferase) with any PET reporter gene(including, but not limited to, HSVI-sr39tk). It should also beappreciated that these reporter genes may be arranged in any order inthe fusion vector, relative to each other.

Moreover, this invention is not limited to the particular methodology,protocols, cell lines, animal species or genera, constructs, andreagents described as such may vary, as will be appreciated by one ofskill in the art. The scope of the invention is, therefore, indicated bythe following claims rather than by the foregoing description. Allchanges, modifications, and variations coming within the meaning andrange of equivalency of the claims are to be considered within theirscope.

1. A double fusion expression vector comprising: a first nucleic acidsequence, wherein the first nucleic acid sequence encodes a polypeptidehaving a first imagable property; and a second nucleic acid sequence,wherein the second nucleic acid sequence encodes a polypeptide having asecond imagable property, and wherein the first and second nucleic acidsequences are linked in frame and are operably linked to an expressioncontrol system.
 2. The double fusion expression vector of claim 1,wherein the first nucleic acid encodes a PET reporter polypeptide. 3.The double fusion expression vector of claim 1, wherein the firstnucleic acid encodes HSV1-sr39 thymidine kinase.
 4. The double fusionexpression vector of claim 3, wherein the first nucleic acid encodeswild type HSV1 thymidine kinase.
 5. The double fusion expression vectorof claim 1, wherein the first nucleic acid encodes a bioluminescentpolypeptide.
 6. The double fusion expression vector of claim 5, whereinthe first nucleic acid encodes renilla luciferase.
 7. The double fusionexpression vector of claim 5, wherein the first nucleic acid encodesfirefly luciferase.
 8. A cultured cell comprising the vector of claim 1.9. A cultured cell transfected with the vector of claim 1, or a progenyof said cell, wherein the cell expresses the double fusion polypeptide.10. A non-human cell comprising the vector of claim
 1. 11. A non-humancell transfected with the vector of claim 1, or a progeny of said cell,wherein the cell expresses the double fusion polypeptide.
 12. A culturedcell comprising: a first nucleic acid sequence, wherein the firstnucleic acid sequence encodes a polypeptide having a first imagableproperty; and a second nucleic acid sequence, wherein the second nucleicacid sequence encodes a polypeptide having a second imagable propertyand is linked in frame to the first nucleic acid sequence and whereinthe first and second nucleic acid sequences are operably linked to anexpression control system.
 13. A non-human cell comprising: a firstnucleic acid sequence, wherein the first nucleic acid sequence encodes apolypeptide having a first imagable property; and a second nucleic acidsequence, wherein the second nucleic acid sequence encodes a polypeptidehaving a second imagable property and is linked in frame to the firstnucleic acid sequence and wherein the first and second nucleic acidsequences are operably linked to an expression control system.
 14. Atriple fusion expression vector comprising: a first nucleic acidsequence, wherein the first nucleic acid sequence encodes a polypeptidehaving a first imagable property; a second nucleic acid sequence,wherein the second nucleic acid sequence encodes a polypeptide having asecond imagable property; and a third nucleic acid sequence, wherein thethird nucleic acid sequence encodes a polypeptide having a thirdimagable property, and wherein the first, second and third nucleic acidsequences are linked in frame and are operably linked to an expressioncontrol system.
 15. The triple fusion expression vector of claim 14,wherein the first nucleic acid encodes a PET repeater polypeptide. 16.The triple fusion expression vector of claim 15, wherein the firstnucleic acid encodes HSV1-sr39 thymidine kinase.
 17. The triple fusionexpression vector of claim 15, wherein the first nucleic acid encodeswild type HSV1 thymidine kinase.
 18. The triple fusion expression vectorof claim 14, wherein the first nucleic acid encodes a bioluminescentpolypeptide.
 19. The triple fusion expression vector of claim 18,wherein the first nucleic acid encodes milk luciferase.
 20. The triplefusion expression vector of claim 18, wherein the first nucleic acidencodes firefly luciferase.
 21. The triple fusion expression vector ofclaim 14, wherein the first nucleic acid encodes a fluorescencepolypeptide.
 22. The triple fusion expression vector of claim 21,wherein the first nucleic acid encodes red fluorescence protein.
 23. Thetriple fusion expression vector of claim 21, wherein the first nucleicacid encodes green fluorescence protein.
 24. A cultured cell comprisingthe vector of claim
 14. 25. A cultured cell transfected with the vectorof claim 14, or a progeny of said cell, wherein the cell expresses thetriple fusion polypeptide.
 26. A non-human cell comprising the vector ofclaim
 14. 27. A non-human cell transfected with the vector of claim 14,or a progeny of said cell, wherein the cell expresses the triple fusionpolypeptide.
 28. A cultured cell comprising: a first nucleic acidsequence, wherein the first nucleic acid sequence encodes a polypeptidehaving a first imagable property; a second nucleic acid sequence,wherein the second nucleic acid sequence encodes a polypeptide having asecond imagable property; and a third nucleic acid sequence, wherein thethird nucleic acid sequence encodes a polypeptide having a thirdimagable property, and wherein the first, second and third nucleic acidsequences are linked in frame and are operably linked to an expressioncontrol system.
 29. A non-human cell comprising: a first nucleic acidsequence, wherein the first nucleic acid sequence encodes a polypeptidehaving a first imagable property; a second nucleic acid sequence,wherein the second nucleic acid sequence encodes a polypeptide having asecond imagable property; and a third nucleic acid sequence, wherein thethird nucleic acid sequence encodes a polypeptide having a thirdimagable property, and wherein the first, second and third nucleic acidsequences are linked in frame and are operably linked to an expressioncontrol system.
 30. A double fusion expression vector comprising: afirst nucleic acid sequence, wherein the first nucleic acid sequenceencodes a polypeptide detectable by PET; and a second nucleic acidsequence, wherein the second nucleic acid sequence encodes abioluminescent polypeptide, and wherein the first and second nucleicacid sequences are linked in frame and are operably linked to anexpression control system.
 31. The double fusion expression vector ofclaim 30, wherein the first nucleic acid encodes HSV1-sr39 thymidinekinase.
 32. The double fusion expression vector of claim 30, wherein thefirst nucleic acid encodes wild type HSV1 thymidine kinase.
 33. Thedouble fusion expression vector of claim 30, wherein the second nucleicacid encodes renilla luciferase.
 34. A cultured cell comprising thevector of claim
 30. 35. A cultured cell transfected with the vector ofclaim 30, or a progeny of said cell, wherein the cell expresses thedouble fusion polypeptide.
 36. A non-human cell comprising the vector ofclaim
 30. 37. A non-human cell transfected with the vector of claim 30,or a progeny of said cell, wherein the cell expresses the double fusionpolypeptide.
 38. A cultured cell comprising: a first nucleic acidsequence, wherein the first nucleic acid sequence encodes a polypeptidedetectable by PET; and a second nucleic acid sequence, wherein thesecond nucleic acid sequence encodes a bioluminescent polypeptide, andwherein the first and second nucleic acid sequences are linked in frameand are operably linked to an expression control system.
 39. A non-humancell comprising: a first nucleic acid sequence, wherein the firstnucleic acid sequence encodes a polypeptide detectable by PET; and asecond nucleic acid sequence, wherein the second nucleic acid sequenceencodes a bioluminescent polypeptide, and wherein the first and secondnucleic acid sequences are linked in frame and are operably linked to anexpression control system.
 40. A double fusion expression vectorcomprising: a first nucleic acid sequence, wherein the first nucleicacid sequence encodes a HSV1-sr39 thymidine kinase; and a second nucleicacid sequence, wherein the second nucleic acid sequence encodes arenilla luciferase, and wherein the first and second nucleic acidsequences are linked in frame and are operably linked to an expressioncontrol system.
 41. A triple fusion expression vector comprising: afirst nucleic acid sequence, wherein the first nucleic acid sequenceencodes a synthetic renilla luciferase; and a second nucleic acidsequence, wherein the second nucleic acid sequence encodes a redfluorescence protein; and a third nucleic acid sequence, wherein thethird nucleic acid encodes a truncated version of HSV1-sr39 thymidinekinase and wherein the first, second and third nucleic acid sequencesare linked in frame and are operably linked to an expression controlsystem.
 42. A noninvasive method for detecting the level of expressionof a gene of interest in an animal using multimodality imaging,comprising administering the double fusion vector of claim 40 to theanimal, wherein the expression control system is the expression controlsystem of the gene of interest, administering coelenterazine to theanimal, immobilizing the animal within the detection field of aphotodetection device, measuring the level of light emission fromrenilla luciferase in the animal with the photodetection device,immobilizing the animal within the detection field of a PET device, andmeasuring the level of positron emission.
 43. A noninvasive method fordetecting the level of expression of a gene of interest in an animalusing multimodality imaging, comprising administering the triple fusionvector of claim 41 to the animal, wherein the expression control systemis the expression control system of the gene of interest, administeringcoelenterazine to the animal, immobilizing the animal within thedetection field of a photodetection device, measuring the level of lightemission from synthetic renilla luciferase in the animal with thephotodetection device, measuring the level of light emission from redfluorescence protein in the animal with the photodetection device,immobilizing the animal within the detection field of a PET device, andmeasuring the level of positron emission.